This invention relates to methods for identifying compounds that modify transcriptional responses to hypoxia.
As solid tumors evolve from a single malignant cell into a multicellular mass, oxygen tension in the tumor microenvironment drops as the passive diffusional capacity of the existing blood supply is surpassed (Nordsmark et al., Acta. Oncol. 33:383–389, 1994; Helmlinger et al., Nat. Med. 3:177–182, 1997; Adam et al., Head Neck 21:146–153, 1999). Tumor hypoxia decreases the efficacy of many common therapies (Brown, Mol. Med. Today 6:157–162, 2000), is a powerful stimulus for angiogenesis (Shweiki et al., Nature 359:843–845, 1992), and is linked to tumor progression (Zhong et al., Cancer Res. 59:5830–5835, 1999; Hockel et al., Cancer Res. 56:4509–4515, 1996) and metastasis (Brizel et al., Cancer Res. 56:941–943, 1996). Since chronic hypoxia differentiates tumor cells from their normal tissue counterparts (Zhong et al., Cancer Res. 59:5830–5835, 1999), therapeutic strategies have been developed that seek to exploit this difference by the design of drugs that are selectively activated under hypoxic conditions (Zeman et al., Int. J. Radiat. Oncol. Biol. Phys. 12:1239–1242, 1986) and hypoxia-induced gene therapy protocols (Binley et al., Gene Ther. 6:1721–1727, 1999; Shibata et al., Gene Ther. 7:493–498, 2000).
Cellular hypoxia triggers a multifaceted adaptive response that is primarily mediated by the heterodimeric transcription factor, HIF-1 (MacDonald et al., Mol. Cell. Biol. 13:5907–5917, 1993; Maltepe et al., Nature 386:403–407, 1997). Under normoxic conditions, the alpha subunit (HIF-1α) is essentially undetectable due to its destruction by the ubiquitin-proteosome system (Salceda et al., J. Biol. Chem. 272:22642–22647, 1997; Huang et al., J. Biol. Chem. 271:32253–32259, 1996). This is achieved, in part, through targeting by the von Hippel-Lindau protein (Huang et al., J. Biol. Chem. 271:32253–32259, 1996). Under hypoxic conditions, through as yet unclear mechanisms, HIF-1α is stabilized and accumulates (Jiang et al., Am. J. Physiol. 271:C1172–1180, 1996). Stabilized HIF-1α heterodimerizes with its binding partner, ARNT (aryl hydrocarbon receptor nuclear translocator) (Wang et al., J. Biol. Chem. 270:1230–1237, 1995; Jiang et al., J. Biol. Chem. 271:17771–17778, 1996), a common binding partner of several βHLH-PAS domain proteins. The heterodimer interacts with p300/CBP (Arany et al., Proc. Natl. Acad. Sci. U.S.A. 93:12969–12973, 1996; Ebert et al., Mol. Cell. Biol. 18:4089–4096,1998; Kallio et al., EMBO J. 17:6573–6586, 1998; Carrero et al., Mol. Cell. Biol. 20:402–415, 2000) and SRC-1 family coactivators (Carrero et al., Mol. Cell. Biol. 20:402–415, 2000), binds to a cognate hypoxia-response element (HRE) (Jiang et al., J. Biol. Chem. 271:17771–17778, 1996), and thereby transactivates HRE-containing promoters and enhancers. Expression of HIF-1 target genes serves to maintain cellular homeostasis, at least in part, by promoting anaerobic glycolysis, facilitating erythropoiesis, and increasing blood delivery through vasodilatation and angiogenesis (Semenza, Annu. Rev. Cell. Dev. Biol. 15:551–578, 1999). The importance of the HIF-1 response pathway in human tumorigenesis is underscored by the finding that HIF-1α is overexpressed in multiple human cancers (Zhong et al., Cancer Res. 59:5830–5835, 1999).
Adaptation to hypoxic conditions not only plays a role in tumor progression, but is also important in conditions of local tissue hypoxia/anoxia, such as in the pathogenesis of myocardial ischemia and stroke. The HIF pathway has also been shown to be important in certain inflammatory conditions, such as Crohn's disease.